Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS4687839 A
Publication typeGrant
Application numberUS 06/812,893
Publication dateAug 18, 1987
Filing dateDec 23, 1985
Priority dateDec 23, 1985
Fee statusLapsed
Publication number06812893, 812893, US 4687839 A, US 4687839A, US-A-4687839, US4687839 A, US4687839A
InventorsTomas G. Kempe
Original AssigneeKempe Tomas G
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Calcitonin gene related peptide analogs with C-terminal D-amino acid substituents
US 4687839 A
Abstract
New calcitonin gene related peptide analogs (CGRP) are disclosed which have biological activity of the same type as known calcitonin gene related peptides and which have a D-amino acid substituent in at least one of the positions 36 and 37 instead of the natural L-amino acids.
Images(5)
Previous page
Next page
Claims(4)
What is claimed is:
1. [D-Ser36 ] human calcitonin gene-related peptide.
2. [D-Thr36 ] human calcitonin gene-related peptide.
3. [D-Asp36 ] human calcitonin gene-related peptide.
4. [D-Asn36 ] human calcitonin gene-related peptide.
Description
FIELD OF THE INVENTION

This invention relates to calcitonin gene related peptide analogs (CGRP) having biological activity and to peptides which can be converted to biologically active CGRP analogs.

BACKGROUND OF THE INVENTION

All known calcitonin gene related peptide ("CGRP") analogs share some common structural features. Each is 37 amino acids long with a C-terminal phenylalanine amide and an N-terminal disulfide linked ring from position 2 through position 7. One of the human CGRP's, for example, has the following formula (Morris, H. R. et al. (1984) Nature 308, 746-748) ##STR1## A second recently characterized human CGRP differs from the above by having the amino acid Asn at position 3, Met at position 22 and Ser at position 25 (Steenbergh, P. H. et al. (1985) Febs Lett. 183, 403-407).

There are also two known CGRP of rat origin. The first to be characterized differs from the human sequence above by having Ser at position 1, Asn at position 3, Asp at position 25 and Glu at position 35 (Amara, S. G. et al. (1982) Nature, 298, 240-244). A recently characterized second rat CGRP differs from the human structure by having Ser at position 1, Asn at position 3, and Asp at position 25 (Amara, S. G. et. al., Science 229, 1094-97, 1985).

It has been shown that CGRP and calcitonin are derived from the same gene. The calcitonin gene is alternatively expressed in a tissue specific fashion producing either the calcium-regulating hormone calcitonin in the thyroid or the neuropeptide CGRP in the brain (Rosenfeld, M. G. et al. (1983) Nature 304, 129-135). Recently, it has been shown that both peptides are produced in thyroid parafollicular cells (Lee, Y. et al. (1985) Neuroscience 15, 1227-1237; Self, C. H. et al. (1985) Peptides 6, 627-630) which indicate a peripheral physiological role. Both calcitonin and CGRP interact with the same receptor in some experimental conditions, thus showing that both peptides share some common structural features (Goltzman, D. and Mitchell, J. (1985) Science 227, 1343-1345; Wohlwend, A. et al. (1985) Biochem. Biophys. Res. Commun. 131, 537-542). The inhibition of gastric acid secretion by both peptides also confirms such similarities (Hughes, J. J. et al, (1984) Peptides 5, 665-667; Stevenson, J. C. (1985); Clin. Endocrinol. 22, 655-660; Lenz, H. J. et. al., (1985) Gut 26, 550-555). CGRPs are potent vasodilators; a property that has not been detected in human calcitonin (Brain, S. D. et al. (1985) Nature 313, 54-56). U.S. Pat. No. 4,530,838 discloses two naturally occurring rat CGRPs and U.S. Pat. No. 4,549,986 discloses human CGRP.

SUMMARY OF THE INVENTION

I have discovered that D-amino acid substituents at the C-terminal portion in synthetic CGRP analogs provide CGRP analogs having biological activity of the same type as known CGRP and calcitonin analogs. In these new peptides, the amino acid sequence contains at least one D-amino acid residue at position 36 or position 37 or both. The new peptides have good potency and quality when compared with known CGRPs. The introduction of D-amino acids may result in increased bioactivity due to increased stability and/or specific structural features of the peptide. I suggest that some of the similarities in biological activities between calcitonins and CGRPs are partly due to the sequence homology at the C-terminal portion of the peptides as shown below: ##STR2## The CGRP analogs may be those of human or rat origin. Preferably, a D-amino acid residue is at position 36 only.

Preferred D-amino acid substituents are D-Ala, D-Val, D-Ile, D-Ser, D-Thr, D-Asp, D-Asn, D-Glu and D-Gln. A preferred analog is substituted human CGRP of the formula: ##STR3## in which X or Y or both are, independently, D-Ala, D-Val, D-Leu, D-Ile, D-Ser, D-Thr, D-Asp, D-Asn, D-Glu, D-Gln, D-Pro, D-Hypro, D-Phe and D-Tyr, or the corresponding L-amino acids, at least one of X or Y being a D-amino acid residue. Particularly preferred peptides of the invention are the human analogs, especially [D-Ser36 ] CGRP, [D-Thr36 ] CGRP, [D-Asp36 ] CGRP [D-Asn36 ] CGRP, [D-Glu36 ] CGRP and [D-Gln36 ] CGRP.

DESCRIPTION OF THE INVENTION

Resin Peptide Synthesis

The synthesis of CGRP analogs may follow the stepwise solid phase strategy reported in Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154, the teachings of which are incorporated herein by reference. The acid labile tert-butyloxycarbonyl (Boc-) group may be used for temporary alpha-N protection and the more acid stable groups may be used for protection of the side chains of the amino acids. Amino acid derivatives are listed in Table 1 and abbreviations are listed in Table 2. Attachment of the peptide chain to a copolymer matrix of styrene and 1% divinylbenzene may employ a benzhydrylamine type "handle" as reported in Pietta, P. G. et al. (1970) Chem. Commun. 650-651; Hruby, V. J. et al. (1977) J. Org. Chem. 42, 3552-3556; and Tam, J. P. et al. (1981) Tetrahedron Lett. 22, 2851-2854, which teachings also are incorporated by reference. All amino acids may be incorporated following a double coupling protocol with some modifications for particular amino acids. .For all reactions, except for arginine and asparagine, the first coupling employs the preformed symmetric anhydride method (Hagenmaier, H. and Frank, H. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 1973-1976) in dichloromethane and the second coupling employs the preformed hydroxybenztriazole ester method (Konig, W. and Geiger, R. (1970) Chem. Ber. 103, 788-798) in dimethyl formamide (DMF). For Boc-Arg(Tos), standard DCC coupling conditions are employed to reduce the risk of lactam formation. The second coupling is done with the active HOBt ester method in DMF. Boc-Asn is exclusively coupled as HOBt esters in DMF to reduce nitrile and amidine formation (Mojsov, S. et al. (1980) J. Org. Chem. 45, 555-560). N-epsilon-(2-Chlorobenzyloxycarbonyl)lysine, Lys(ClZ), is used because it is more stable than the benzyloxycarbonyl derivative to the acid deprotection steps and it also avoids side chain branching (Erickson, B. W. and Merrifield, R. B. (1972) J. Am. Chem. Soc. 95, 3757-3763). The beta-cyclohexyl ester (cHex) of Boc-Asp-OH is used; it is also more stable to acids and thus minimizes aspartimide formation (Tam, J. P. (1979) Tetrahedron Lett. 4033-4036). The quantitative ninhydrin reaction is routinely used throughout the synthesis to monitor the extent of coupling after each cycle (Sarin, V. K. et al. (1981) Anal. Biochem. 117, 147-157).

              TABLE 1______________________________________Amino acid derivatives for synthesisof CGRP analogs at position 36.[D-Ser36 ] human CGRPcycl nr.                                couplingand     protected amino                 pro-amino acid   acids       MW       mmol  g    cedure______________________________________37      Phe-benzhydryl       1     2    A   amine resin36      Boc-D-Ser(Bzl)               see cycle               34 below34, 19, 17   Boc-Ser(Bzl)               295.1    8     2.36 A                        4     1.1835, 24  Boc-Lys(Cl--Z)               314.8    8     2.50 A                        4     1.2533, 21, 20,   Boc-Gly     175.2    8     1.40 A14                           4     0.7032, 28, 23,   Boc-Val     217.1    8     1.74 A22, 8                        4     0.8731, 26, 25   Boc-Asn     232.2    4     0.93 B30, 9, 6, 4   Boc-Thr(Bzl)               309.1    8     2.48 A                        4     1.2429      Boc-Pro     215.1    8     1.72 A                        4     0.8627      Boc-Phe     265.2    8     2.12 A                        4     1.0618, 11  Boc-Arg(Tos)               442.5    4     1.77 C16, 15, 12   Boc-Leu     249.2    8     2.0  A                        4     1.013, 5, 1   Boc-Ala     189.2    8     1.51 A                        4     0.7610      Boc-His(Tos)               409.2    8     3.28 A                        4     1.647, 2    Boc-Cys     325.2    8     2.60 A   (4-Me Bzl)           4     1.403       Boc-Asp     328.4    8     2.63 A   (OcHex)              4     1.31______________________________________

              TABLE 2______________________________________ Abbreviations (Biochem Biophys. Acta 133, 1-5 (1967)).______________________________________Boc =      tert-butyloxycarbonylBzl =      benzylTos =      tosylCl2 Bzl =      2,6-dichlorobenzylCl--Z =    o-chlorobenzyloxycarbonylOcHex =    gamma-cyclohexyl ester4-Me--Bzl =      4-methylbenzylHOBt =     N--hydroxybenztriazoleDIEA =     diisopropylethylamineDCC =      dicyclohexylcarbodiimideDMF =      N,N--dimethylformamideCM =       carboxymethylTFA =      trifluoroacetic acidHPLC =     high performance liquid chromatographyAla =      L-alanylPro =      L-prolylSer =      L-serylHse =      L-homoserylGly =      glycylThr =      L-threonylAsn =      L-asparaginylArg =      L-arginylTyr =      L-thyronylPhe =      L-phenylalanylLeu =      L-leucylLys =      L-lysylHis =      L-histidylAsp =      L-asparagylVal =      L-valylCys =      L-cysteinylD-Ser =    D-serylD-Thr =    D-threonylD-Asn =    D-asparaginylMet =      L-methionylGlu =      L-glutamylD-Asp =    L-asparagylD-Ala =    D-alanylD-Val =    D-valylD-Ile =    D-isoleucylD-Glu =    D-glutamylD-Gln =    D-glutaminylD-Pro =    D-prolylD-Hypro =  D-hydroxyprolylD-Phe =    D-phenylalanylD-Tyr =    D-thyronyl______________________________________

Resin Peptide Cleavage and Purification

Cleavage of the peptides from the resin and removal of all the remaining protecting groups is accomplished by treatment with anhydrous hydrogen fluoride in the presence of anisole (Yamashiro, D. and Li, C. H. (1978) J. Am. Chem. Soc. 100, 5174-5179). Crude peptide is removed from the resin by washing with 10% aqueous acetic acid. After lyophilization, the residue may be treated with dithiothreitol (Cleland, W. W. (1964) Biochemistry 3, 480-482) in sodium phosphate buffer at pH 7.5. The intramolecular disulfide bond in CGRP between cysteine residues 2 and 7 can be formed by diluting the solution several-fold and adding potassiumferricyanide in aqueous solution. The resultant peptide solution is then con- centrated by passing it through a CM-Sephadex, C-25 column and then eluting with a linear gradient of sodium chloride from zero to 0.3 molar in the same phosphate buffer (Live, D. H. et al. (1977) J. Org. Chem. 42, 3556-3561; Moe, G. R. and Kaiser, E. T. (1985) Biochemistry 24, 1971-1976). The sample is finally desalted by gel filtration, concentrated and isolated by HPLC.

While the D-amino acid substitutions in at least one of the positions 36 or 37 may be made in human CGRP and rat CGRP, for exemplification, the following detailed disclosure is directed to human CGRP. The formula for our new substitution analogs at position 36 and position 37 of human CGRP may be written as follows: ##STR4## in which X is D-Ala, D-Val, D-Leu-, D-Ile, D-Thr, D-Asp, D-Asn, D-Glu and D-Gln, or the corresponding L-amino acids; and Y D-Pro, D-Hypro, D-Phe and D-Tyr or the corresponding L-amino acids. At least one of X and Y is a D-amino acid residue.

As may be seen from the formula above, 37 amino acids are involved and in this formula, the positions are numbered according to the accepted procedure beginning at position 1 for the Ala on one end of the chain and ending with Phe at position 37 at the other end of the chain. For clarity of description, this same numbering system will be followed in referring to the cycles of the synthesis. The assembly of the amino acids begins with cycle 36 which involves the coupling of the amino acid to the Phe moiety, followed by residue 35 and so on to the last amino acid. Protected amino acid derivatives that may be used in the synthesis of CGRP analogs are given in Table 1. The resin which is functionalized with Phe is available from chemical supply houses.

As indicated earlier, three types of coupling procedures are used, depending on the properties of reactants. In Table 1, the amino acid position and cycle number, type of coupling procedure, molecular weights and amount of reactants for the cycle are given. The details for each coupling protocol A, B and C are described below.

RESIN PEPTIDE SYNTHESIS EXAMPLE 1

[D-Ser36 ]CGRP: Double coupling protocol using symmetric anhydride and active ester methods may be used to ensure as complete coupling as possible. The following protocol may be used for all amino acids except for arginine and asparagine. The protocol is given for 2 g benzhydryl type resin functionalized with a total of 1 mMol of Phe.

Coupling Procedure A.

1. The resin is washed with dichloromethane, CH2 Cl2, (30 ml, 61 min).

2. Removal of the Boc protecting group is done with 50% TFA in CH2 Cl2 (30 mL, 31 min) and with 30 mL for 20 min.

3. The reagent is then removed with CH2 Cl2 wash (30 mL, 61 min).

4. Traces of acid are finally removed with 5% DIEA in CH2 Cl2 (30 mL, 22 min).

5. A final wash is done before the coupling is completed, CH2 Cl2 (30 mL, 61 min).

6. 5 mg of the resin are removed for ninhydrin test.

7. The protected amino acid (listed in Table 1, 8 mMol) dissolved in 10 mL of CH2 Cl2 is treated with DCC (4 mMol, 825 mg) in 3 mL of CH2 Cl2. After 10 min, the solution is filtered and added to the resin. The precipitate is washed with 10 mL of CH2 Cl2 and added to the reaction vessel which is then shaken for 2 h at room temperature.

8. The resin is washed with CH2 Cl2 (30 mL, 42 min).

9. The resin is washed with 5% DIEA in CH2 Cl2 (30 mL, 2 min).

10. The resin is washed with CH2 Cl2 (30mL, 42 min).

11. Ninhydrin test is performed.

12. The resin is washed with DMF (30 mL, 22 min).

13. HOBt (4 mMol, 540 mg) in 7 mL of DMF at 0 C. is mixed with DCC (4 mMol, 825 mg) in 3 mL CH2 Cl2. The protected amino acid (listed in Table 1, 4 mMol) dissolved in 6mL of DMF is then added. The mixture is kept for 10 min at 0 C. and is then added to the resin. The mixture is shaken for 2 h at room temperature.

14. The resin is then washed with DMF (30 mL, 22 min).

15. The resin is washed with CH2 Cl2 (30 mL, 41 min).

16. The resin is washed with 5% DIEA in CH2 Cl2 (30 mL, 2 min).

17. The resin is washed with CH2 Cl2 (30 mL, 31 min).

18. Ninhydrin test is performed.

Coupling Procedure B. (Used for the amino acids asparagine):

Steps 1-6 were the same as coupling procedure A.

7. The resin is washed with DMF in CH2 Cl2 (1:2 v/v, 30 mL, 22 min).

8. To HOBt (4 mMol, 540 mg) in 7 mL DMF/CH2 Cl2 (1:1 v/v) at 0 C. is added DCC (4 mMol, 825 mg) in 3 mL of CH2 CL2. To that mixture is then added the protected amino acid (listed in Table 1, 4 mMol) in 6 mL of DMF/CH2 Cl2. The reaction mixture is added to the resin after 10 min at 0 C. The resin is then shaken for 2 h at room temperature.

9. The resin is washed with DMF/CH2 Cl2 (1:2 v/v, 30 mL, 22 min).

The steps 8-18 described in coupling procedure A are then followed.

Coupling Procedure C. (Used for the amino acid arginine):

Steps 1-6 are the same as coupling procedure A.

7. The protected amino acid (listed in Table 1, 4 mMol) in 10 mL CH2 Cl2 is added to the resin. DCC (4 mMol, 825 mg) in 3 mL CH2 Cl2 is added after 5 min to the resin. The reaction mixture is then shaken for 2 h at room temperature.

The steps 8-18 described in coupling procedure A are then followed.

EXAMPLE 2

[D-Thr36 ]CGRP: Boc-D-Thr(Bzl) is used in cylce 36, and coupling procedure A is employed. The preceding couplings were the same as previously described (Table 1).

EXAMPLE 3

[D-Asn36 ]CGRP: Boc-D-Asn is used in cycle 36, and coupling procedure B is employed. The preceding couplings were the same as previously described (Table 1).

In each example, the addition of Ala1 represents the completion of the solid phase synthesis. The Boc group is finally removed by steps 1-6 in coupling procedure A. The resin peptides are then removed from the reaction vessel and dried in vacuum. Cleavage and purification steps are carried out as follows:

RESIN-PEPTIDE CLEAVAGE

The dried resin peptide (2 g ) and 2 mL of anisole are placed in a teflon reaction vessel which is cooled in a dry ice-acetone bath and about 15 mL of hydrogen fluoride gas is condensed into the vessel. The mixture is stirred at 0 C. in an ice bath for 45 min. The hydrogen fluoride is then evaporated under vacuum, using first a water aspirator and later a high vacuum pump. The residue is triturated with 530 mL of ethyl acetate, and the peptide was extracted from the resin beads with 100 mL of 10% aqueous acetic acid solution. The mixture was lyophilized to dryness.

PURIFICATION OF CRUDE PEPTIDES

A 100 mg sample of the lyophilized peptide is treated with excess dithiothreitol (5 mMol) in 5 mL of 50 mM sodium phosphate buffer at pH 7.5 for 1 h at room temperature. The intramolecular disulfide bond between cysteine residues 2 and 7 is formed by diluting the peptide solution to a volume of 1 liter in the same buffer. A solution of 20 mM K3 Fe(CN)6 is slowly added with stirring until a persistant yellow color is obtained. The resultant dilute peptide solution is concentrated by passing it through a CM-Sephadex, C-25 column and then eluting with a linear gradient of NaCl from zero to 0.3M employing the same buffer. Fractions from this column may be desalted on a Sephadex G-15 column, eluting with a 0.03M aqueous acetic acid solution. Samples for biological testing are isolated on an analytical HPLC (column: Altex ODS, 5 micron, 4.6250 mm, flow 1.5 mL/min, gradient of 30-45% acetonitrile in 0.1M ammonium acetate buffer at pH 5.5).

The HPLC isolated samples are hydrolyzed with 5.5M hydrochloric acid, and amino acid analyses are performed to confirm the chemical composition. The new polypeptides are biologically active. They exhibit similarities to both known calcitonins and CGRPs in lowering gastric acid secretion.

While only certain embodiments of my invention have been described in specific details, it will be apparent to those skilled in the art that many other specific embodiments may be practiced and many changes may be made, all within the spirit of the invention and the scope of the appended claims.

Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US3929758 *Sep 12, 1974Dec 30, 1975Armour PharmaCyclization of cysteine-containing peptides
US4530838 *Jul 8, 1983Jul 23, 1985The Salk Institute For Biological StudiesSynthetic calcitonin-gene-related peptides for lowering blood pressure or gastric acid secretion in mammals
US4549986 *Dec 23, 1983Oct 29, 1985The Salk Institute For Biological StudiesHypotensive and antisecretory agent
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US5175145 *Jan 14, 1992Dec 29, 1992Amylin Pharmaceuticals, Inc.Treatment of diabetes mellitus with amylin agonists
US5266561 *Jun 4, 1991Nov 30, 1993Amylin Pharmaceuticals, Inc.Treatment of type 2 diabetes mellitus
US5281581 *Jun 19, 1992Jan 25, 1994Amylin Pharmaceuticals, Inc.Treatment of insulin resistance
US5641744 *Nov 17, 1994Jun 24, 1997Amylin Pharmaceuticals, Inc.Treatment of diabetes mellitus
US5716619 *Aug 23, 1994Feb 10, 1998Amylin Pharmaceuticals, Inc.Treatment of type 2 diabetes mellitus
US5942227 *Jan 11, 1996Aug 24, 1999Amylin Pharmaceuticals, Inc.Treating type 2 diabetes mellitus or insulin resistance patient
US6743429Dec 30, 1999Jun 1, 2004Sherbrooke UniversityUse of calcitonin gene-related peptide in the prevention and alleviation of asthma and related bronchospastic pulmonary diseases
US8076288Aug 17, 2005Dec 13, 2011Amylin Pharmaceuticals, Inc.Hybrid polypeptides having glucose lowering activity
US8168592Oct 19, 2006May 1, 2012Amgen Inc.CGRP peptide antagonists and conjugates
US8263545Aug 17, 2007Sep 11, 2012Amylin Pharmaceuticals, Inc.GIP analog and hybrid polypeptides with selectable properties
US8404637Feb 10, 2006Mar 26, 2013Amylin Pharmaceuticals, LlcGIP analog and hybrid polypeptides with selectable properties
US8497240Aug 17, 2007Jul 30, 2013Amylin Pharmaceuticals, LlcDPP-IV resistant GIP hybrid polypeptides with selectable properties
US8697647Dec 12, 2011Apr 15, 2014Odile Esther LevyHybrid polypeptides with selectable properties
DE3913954A1 *Apr 27, 1989Oct 31, 1990Stief GeorgArzneimittel zur behandlung erektiler dysfunktionen
EP2248527A2Mar 31, 2006Nov 10, 2010Amylin Pharmaceuticals, Inc.Compositions and methods for the control, prevention, and treatment of obesity and eating disorders
EP2330124A2Aug 11, 2006Jun 8, 2011Amylin Pharmaceuticals Inc.Hybrid polypeptides with selectable properties
EP2330125A2Aug 11, 2006Jun 8, 2011Amylin Pharmaceuticals, Inc.Hybrid polypeptides with selectable properties
EP2390264A1Feb 10, 2006Nov 30, 2011Amylin Pharmaceuticals Inc.GIP analog and hybrid polypeptides with selectable propperties
EP2392595A1Feb 10, 2006Dec 7, 2011Amylin Pharmaceuticals Inc.GIP analog and hybrid polypeptides with selectable properties
EP2417980A1Feb 11, 2005Feb 15, 2012Amylin Pharmaceuticals Inc.Hybrid polypeptides with selectable properties
EP2422806A2Feb 11, 2005Feb 29, 2012Amylin Pharmaceuticals Inc.Hybrid polypeptides with selectable properties
EP2422807A2Feb 11, 2005Feb 29, 2012Amylin Pharmaceuticals Inc.Hybrid polypeptides with selectable properties
WO1992015695A1 *Mar 2, 1992Sep 2, 1992Carlbiotech Ltd AsProcess for the c-terminal modification of peptides having a c_terminal penultimate proline residue
Classifications
U.S. Classification530/324, 930/DIG.672, 930/60, 930/21, 930/20
International ClassificationC07K14/585
Cooperative ClassificationC07K14/5855
European ClassificationC07K14/585A
Legal Events
DateCodeEventDescription
Oct 31, 1995FPExpired due to failure to pay maintenance fee
Effective date: 19950823
Aug 20, 1995LAPSLapse for failure to pay maintenance fees
Mar 28, 1995REMIMaintenance fee reminder mailed
Mar 19, 1991REMIMaintenance fee reminder mailed